Enhanced expression of the beta II subspecies of protein kinase C in differentiating erythroleukemia cells

Polyclonal antipeptide antibodies which recognize selected isozymes (alpha, beta I, beta II, and gamma) of the protein kinase C family were used to identify specific subspecies in undifferentiated Friend erythroleukemia cells and in cells triggered to differentiate with hexamethylene bisacetamide. The beta II isozyme of protein kinase C was the primary isozyme expressed and its abundance was significantly increased (P less than 0.05) in differentiated cells. Differences in immunostaining between control and experimental groups were objectively quantitated by determining percentage transmission of light through cells based on color threshold rather than gray intensity levels. Staining was localized to the cytoplasm predominantly in differentiated cells, whereas nuclei stained more intensely in undifferentiated cells. These results provide immunocytochemical evidence to support the hypothesis that changes in the expression of the beta II subspecies of protein kinase C are essential to the programmed maturation of differentiating Friend erythroleukemia cells.     

15-Lipoxygenase products affect protein phosphorylation in Friend erythroleukemia cells

Endogenous arachidonic acid metabolism and protein phosphorylation have been examined in Friend erythroleukemia cells in response to the induction of differentiation by dimethyl sulfoxide and hexamethylene bisacetamide. 15-Hydroxyeicosatetraenoic acid levels were elevated in cells differentiated with hexamethylene bisacetamide or dimethyl sulfoxide compared with undifferentiated cells. Protein phosphorylation decreased markedly in differentiated cells compared with undifferentiated cells and the addition of 15-hydroperoxyeicosatetraenoic acid specifically decreased the phosphorylation of a 28-kilodalton protein. These findings indicate that products of 15-lipoxygenase may act as intracellular messengers in Friend erythroleukemia cells by affecting protein phosphorylation.     

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.     

Prostaglandin A1 induces differentiation in friend erythroleukemia cells

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B₂ and 6-keto PGF₁α, were completely inactive, while PGE₁ inhibited slightly and PGF₂α stimulated the replication of FLC. PGA₁ was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA₁ alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA₁-stimulated differentiation was completely suppressed by the addition of 10⁻⁶M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA₁ (but no DMSO) prevented the return to the undifferentiated state.     

Differential inhibition by α- and β-tocopherol of human erythroleukemia cell adhesion: role of integrins

The effect of α- and β-tocopherol on human erythroleukemia cell (HEL) adhesion induced by phorbol 12-myristate 13-acetate (PMA) has been studied. Adhesion induced by PMA stimulation was prevented by 44.5% by physiological concentrations of α-tocopherol. Under the same experimental conditions, β-tocopherol, an analogue of α-tocopherol, produced 11% inhibition of adhesion. Cell response gradually increased from 0 to 24 h of α-tocopherol treatment. Only a slight time dependency of β-tocopherol inhibition was observed. Another human erythroleukemia cell line (K562) and the human monocyte tumor cell line U937 showed 5.0 and 11.2% inhibition, respectively. Similar to α-tocopherol, the protein kinase C inhibitor, Calphostin C, and the MAPK inhibitor, PD98059, prevented PMA-induced cell adhesion. An inhibition of ERK-1 phosphorylation was observed for α-tocopherol only in HEL, implying that MAP kinase pathway is involved in this cell line. Fluorescence-activated cell sorting (FACS), by using various integrin-specific monoclonal antibodies, has shown that α (1–6), β1, and αv integrins are less expressed at the cell surface after α-tocopherol treatment. Beta-tocopherol treatment was less effective.     

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [¹⁴C] glycerol or [³H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.     

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β-1, β-2, β-3, β-4 and β-5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.     

Phosphatidylinositol-3-kinase activation and atypical protein kinase C zeta phosphorylation characterize the DMSO signalling in erythroleukemia cells

Here we provide evidence for a role of phosphatidylinositol-3-kinase (PI-3-kinase) and for its product phosphatidylinositol-3,4, 5-triphosphate (PI3,4,5P3) in the occurrence of the metabolic differentiation state induced by DMSO in murine Friend erythroleukemia cells. Of note, the activation of PI-3-kinase correlated with the modulation of the activation of another enzyme, the atypical protein kinase C zeta (aPKC zeta). In particular, the expression of PI-3-kinase was substantially unaffected by DMSO treatment while its phosphorylation and the production of PI3,4,5P3 was strongly increased within 24 h of DMSO. Such a result was paralleled by an evident phosphorylation of a PKC zeta. Treatment of the cells with the two unrelated PI-3-kinase inhibitors wortmannin and LY 294002 impaired the recovery of the number of differentiated cells, therefore indicating that PI-3-kinase might be involved in the induction of erythroid differentiation, possibly engaging a protein kinase C zeta as downstream effector.     

Sulfhydryl groups and the differentiation of murine erythroleukemia cells.

The importance of cysteine and sulfhydryl groups has been demonstrated in relation to the differentiation and respiration of Friend erythroleukemia cells (FLC). The respiratory rate of undifferentiated FLC was higher basally (5.06 ± 0.16 vs. 3.10 ± 0.09 nmoles 02/min/10⁶ cells) and was further 70% stimulated by addition of cysteine, whereas DMSO-induced differentiated cells were insensitive. A sulfhydryl blocking agent (PCMS) was capable of maintaining the differentiated state of FLC cultured in the absence of DMSO and this effect appeared to be reversible upon removal of the PCMS.     

EB1 Levels Are Elevated in Ascorbic Acid (AA)-stimulated Osteoblasts and Mediate Cell-Cell Adhesion-induced Osteoblast Differentiation

Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I α1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of β-catenin distribution at the cell cortex and within the nucleus. Diminished β-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-β-catenin and active glycogen synthase kinase 3β, a kinase known to target β-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the β-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with β-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and β-catenin. We also determined that cell-cell contacts and cortically associated EB1/β-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.     

Regulation of the Cell Cycle at the G2/M Boundary in Metastatic Melanoma Cells by 12-O-Tetradecanoyl Phorbol-13-acetate (TPA) by Blocking p34Kinase Activity

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppocket al.,1992,Cell Growth Differ.3, 485–494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34^cdc2/cyclin B1 kinase activity. In cells treated with TPA, most p34^cdc2was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21^Cip1/Waf1, but not of p27^Kip1, was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC α, βI, βII, δ, ε, ι/λ, ζ, and μ isozymes. PKC η and PKC θ were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC α, βI, βII, δ, and ε isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC δ appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC μ was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34^cdc2kinase activity which is associated with the increased expression of p21^Cip1/Waf1and increased phosphorylation on tyrosine of p34^cdc2. This arrest, in turn, is associated with a shift of PKC isozymes PKC α, PKC βI, PKC βII, PKC δ, PKC ε, and PKC μ to the membrane fraction which is induced by addition of TPA.     

Activation of the Jun N-terminal kinase pathway by friend spleen focus-forming virus and its role in the growth and survival of friend virus-induced erythroleukemia cells

Members of the mitogen-activated protein kinase (MAPK) family, including Jun amino-terminal kinase (JNK) and extracellular signal-related kinase (ERK), play an important role in the proliferation of erythroid cells in response to erythropoietin (Epo). Erythroid cells infected with the Friend spleen focus-forming virus (SFFV) proliferate in the absence of Epo and show constitutive activation of Epo signal transduction pathways. We previously demonstrated that the ERK pathway was constitutively activated in Friend SFFV-infected erythroid cells, and in this study JNK is also shown to be constitutively activated. Pharmacological inhibitors of both the ERK and JNK pathways stopped the proliferation of primary erythroleukemic cells from Friend SFFV-infected mice, with little induction of apoptosis, and furthermore blocked their ability to form Epo-independent colonies. However, only the JNK inhibitor blocked the proliferation of erythroleukemia cell lines derived from these mice. The JNK inhibitor caused significant apoptosis in these cell lines as well as an increase in the fraction of cells in G(2)/M and undergoing endoreduplication. In contrast, the growth of erythroleukemia cell lines derived from Friend murine leukemia virus (MuLV)-infected mice was inhibited by both the MEK and JNK inhibitors. JNK is important for AP1 activity, and we found that JNK inhibitor treatment reduced AP1 DNA-binding activity in primary erythroleukemic splenocytes from Friend SFFV-infected mice and in erythroleukemia cell lines from Friend MuLV-infected mice but did not alter AP1 DNA binding in erythroleukemia cell lines from Friend SFFV-infected mice. These data suggest that JNK plays an important role in cell proliferation and/or the survival of erythroleukemia cells.     

Quantitative Comparison of Constitutive Promoters in Human ES cells

Background

Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking.

Methodology/Principal Findings

We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies.

Conclusion/Significance

The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75–80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.     

Actions of lipoxins A4 and B4 on signal transduction events in Friend erythroleukemia cells.

Earlier studies in our laboratory suggested a role for 15-lipoxygenase products of arachidonic acid, such as 15-hydroperoxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid, in supporting proliferative events in Friend erythroleukemia cells. Because lipoxins are also products of the same lipoxygenase enzyme, we tested their actions on signal transduction events related to DNA synthesis. Lipoxins A4 and B4 (10 nM) significantly enhanced [3H]thymidine incorporation into Friend cells in the absence of fetal bovine serum without affecting cell differentiation or cell number. Lipoxin B4 increased the duration of time that cells spent in the S phase of the cell cycle, and also significantly enhanced protein kinase C activity in nuclei, whereas c-fos expression was unaffected by either of the lipoxins tested. The novel, intracellular actions of lipoxins A and B on Friend erythroleukemia cells documented in this study represent a unique spectrum of effects of lipoxins on signal transduction events as compared with other eicosanoids.     

Regulation of β-adrenergic responses during in vitro differentiation of mouse erythroleukemia cells

Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in β-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the β-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor—cyclase coupling is probably due to a major shift in the expression of G_i and G_s regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is G_i (G_sα:G_iα = 1:7). By contrast, G_s predominates in differentiated cells (G_sα:G_iα = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Minimal CK2 activity required for yeast growth

Protein kinase CK2 is essential for the growth of Saccharomyces cerevisiae. Yeast cells that lack the functional genes coding for both the catalytic subunits of protein kinase CK2 can grow only if they are complemented by exogenous cDNAs coding for this subunit. A series of deletion mutants of CK2α from Xenopus laevis was constructed. These mutants that have carboxyl end deletions yield a CK2α product that varies over four orders of magnitude in its capacity to phosphorylate casein in vitro. Complementation of yeast RPG41-1a, a mutant defective in CKA1 and CKA2 genes, with wild-type X. laevis CK2α and with cDNAs coding for truncated CK2α having amino acids 1–328 and 1–327 resulted in cells that grew in gal-minimal media at 30 ^∘C as well as the cells harboring the yeast CKA2 gene. However, the growth was significantly diminished when cells were complemented with X. laevis CK2α containing 1–326 amido acids. This mutant has 0.6% of the catalytic activity of the wild-type enzyme. Yeast cells that expressed CK2α 1–324 and 1–323 which have 10-and 100-fold less activity, respectively, were not able to grow. The growth of cells containing the CK2α 1–326 mutant was very sensitive to temperature, and minimal growth was observed at 37 ^∘C. This mutant was also more sensitive to UV radiation but was not significantly affected by 0.4 M NaCl.Both authors contributed equally to this work     

Terminal differentiation in cultured Friend erythroleukemia cells.

Two populations of differentiated, hemoglobin-containing cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the G1 phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation.We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in G1 by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in G1. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in G1 after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in G1 with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-C1-2.